Triplex forming Triplex forming oligonucleotidesoligonucleotides

(TFO) (TFO)

Dr Pupak Derakhshandeh, PhD

Ass Prof of Medical Science of Tehran University

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IntroductionIntroduction

DNA structure is a critical element in determining its function

• Agents for modifying gene function

• In most instances they are utilized for repression of transcription

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TFOsTFOs

TFOs can bind in the major groove of DNA:

polypurine / polypyrimidine sequences

forming specific Hoogsteen hydrogen bonds

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Classification of DNA triple helices

• Intermolecular triplexes

• Interamolecular triplexes

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DNA triplex structures

• Either intermolecular triplexes formed by binding of an exogenously applied (TFO, therapeutic)

• Or naturally occurring intra molecular triplexes (H-DNA)

– specific alteration of the genome

– site-specific mutagenesis, for stimulat-

– Ing DNA repair or recombination

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Such that elements of the H-DNA structure may be pharmacologically exploitable

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An example of triplex formation with a poly purine TFO sequence specific

for the human c-MYC P2 promoterThe TFO is placed in an anti parallel

orientation relative to the target duplex

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Canonical base triplets formed in purine and pyrimidine triplex motifs

Watson-Crick base pairing is illustrated by dotted lines

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• In the anti parallel, purine motif:

–G:G-C

–A:A-T

–In the parallel, pyrimidine motif:– T:A-T

– C:G-C

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triplex motifs• Triplex formation is kinetically slow

compared to duplex annealing

• However, once formed, triplexes are very Stable

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Triplex-forming oligonucleotides (TFOs)

Bind DNA in a sequence-specific manner at polypurine / polypyrimidine sites

Mediate targeted genome modification Formation in cells, leading to

mutagenesis or recombination

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The anti-gene and The anti-gene and antisense applicationantisense application

of TFOof TFO

promise of therapeutic utility

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Triplex formationTriplex formation

Triplex formation has been shown to inhibit transcription in mammalian cells

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TFOTFOThey have been used to deliver DNA reactive conjugates to specific target sites:

– leading to site-directed mutagenesis in some cases:• both in mammalian cells • in culture / in vitro even

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It is interesting to note:It is interesting to note:

The hairpin-TFO is able to invade the duplex:

that is present as nucleosome associated chromatin

mutagenesis or gene silencing

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Potential applications of DNA triplex formation in therapeutics

• Allowing the covalent attachment of DNA damaging agents

• A potential advantage of targeting DNA rather than RNA or protein:

– Limited number of copies to be targeted

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Another advantage of targeting DNA rather than RNA or protein:

• Facile synthesis of the reagents

• The availability of a variety of chemical modifications:

– To the bases, sugar-phosphate backbone, and the 5’ and 3’ ends)

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Therapeutic applications of TFO

To silence gene expression

Through antigene approach have been reported in the literature

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Modulating gene expression via triplex formation

• TFOs:

–Act as ‘‘decoy’’ oligonucleotides

–Bind transcription factors , they are not available to bind their duplex consensus sequences for transcription activaion !

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TFOTFO

The up regulation of the The up regulation of the gene (gene (induced mutagenesis ))

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TriplexTriplexformationformation

Is known to induce mutagenesisi.g.:

Activation of human gamma-globin gene expression via triplex-forming oligonucleotides

Mutations in the gamma-globin gene 5 flanking region.

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The up regulation of γ -globin

reduce the symptoms of sickle cell anemia and thalassemia

TFO-directed mutagenesis of the upstream sequences

Xu XS et al. Gene 242: 219–228, 2000

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• the presence of high levels of fetal and -thalassemia, are very common genetic diseases

• when γ-globin genes are Human -globin disorders, such as sickle cell anemia highly expressed

• hemoglobin (HbF, γ) in erythrocytes (~20–30%) that affect over a million people worldwide and cause:– can compensate for the defective -

globin gene product

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• when γ-globin genes are highly expressed:

– the presence of high levels of fetal hemoglobin (HbF, γ) in erythrocytes (~20–30%)can compensate for the defective -globin gene product

– and such patients have a much improved disease condition (Stamatoyannopoulos et al., 1994).

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• In adults, the β-globin gene is predominantly expressed (98%) while the γ-globin gene is expressed at very low levels (<1%).

• To increase the levels of HbF in patients with sickle cell / Beta thalassmia disease:

– many drugs have been developed:

• Butyric acid and its analogs have been found to increase the levels of HbF

• Hydroxyurea

– However, many patients cannot achieve increased HbF with these treatments!

– With hydroxyurea treatment, for example, only about 60% of patients were found to have increased HbF in their erythrocytes

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• Hereditary Persistence of Fetal Hemoglobin (HPFH) is a human genetic condition in which the γ-globin genes continue to be expressed at very high levels in the adult life of individuals.

• single base mutations or a small deletion in the 5′ flanking region of the γ-globin gene

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• Molecular biology studies suggest that most of these mutations are located in binding motifs for transcription regulators such as Sp1, GATA-1, and CP1 site.

• Some mutations also create new binding sites for transcription regulators

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Octamer-binding transcription factor-1 gene

• activating the γ-globin gene expression by triplex-forming oligonucleotide (TFO)-directed targeted mutagenesis

• TFO designed to bind to a site overlapping with an Oct-1 binding site (at the −280 region of the γ-globin gene)

• targeted mutagenesis of the Oct-1 binding site has been achieved by:– transfecting the in-vitro-formed plasmid-oligo

complex into human normal fibroblast (NF) cellsXu XS et al. Gene 242: 219–228, 2000

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• The mutation frequency at the target site was estimated to be 20% by direct DNA sequencing analysis

Xu XS et al. Gene 242: 219–228, 2000

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• In-vivo gene expression assays:

–activation of γ-globin gene expression from these mutations in mouse erythroleukemia (MEL) cells

–The levels of the γ-globin gene expression increased by as much as fourfold in mutants with single base changes

Xu XS et al. Gene 242: 219–228, 2000

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• mutations at the Oct-1 binding site can lead to activation of the γ-globin gene and generate the hereditary persistence of fetal hemoglobin (HPFH) condition

Xu XS et al. Gene 242: 219–228, 2000

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• Using TFOs :– to bind to the γ-globin gene −280 region

• region Oct-1 binding site was achieved in a plasmid construct upon in-vitro triplex formation

• transfection into human cells• the mutation frequency of the target site was

found to be in the range of 20%• The mutations were found to result in

reduced binding of Oct-1 transcription factor to the site

• The mutations also led to γ-globin gene expression in MEL cells

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• triplex-mediated targeted mutagenesis (TFO), oligo Gamma 2, directed targeted mutagenesis of pUSAG9 plasmid was studied

• The pUSAG9 plasmid DNA was incubated with oligo Gamma 2 (10 μM) in triplex binding buffer

• for 2 h at 37°C to induce triplex formation

• The plasmid-oligo complex was transfected into human normal fibroblast (NF) cells

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• Plasmid DNA was isolated from individual colonies and the sequence analyzed

• A total of one hundred plasmids were directly sequence-analyzed and 20 of them were found to contain mutations )−287 to −285 of Aγ-globin gene)

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Oct-1 binding site at the −280 region of the γ-globin gene negatively regulates γ-globin gene expression

mutations in the Oct-1 binding site lead to activation of the γ-globin gene expression

Since TFO-directed targeted mutagenesis has already been demonstrated in mammalian cells

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The sequence of the hairpin-TFO and a potential interaction The sequence of the hairpin-TFO and a potential interaction of the hairpin TFO, with the target duplex and GAL4 proteinof the hairpin TFO, with the target duplex and GAL4 protein

Ghosh,M K, et al. Molecular and Cellular Biochemistry 278: 147–155, 2005

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A bifunctional hairpin-TFO

–including the targeting sequences

–polypurine stretch –genes in Saccharomyces

cerevisiae –could bind GAL4 protein with

high affinity – stable triplex with target

sequence

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The potential use of The potential use of chimaericchimaeric

hairpin-TFO to promote hairpin-TFO to promote transcription activationtranscription activation

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Transcriptional activationTranscriptional activationTriplex forming oligonucleotides +The cognate binding site for transcription

activator Could be targeted to the upstream

poly(pu/py) region of specific genes in vivoLeading to transcriptional activation By endogenously available transcription

activator

Ghosh,M K, et al. Molecular and Cellular Biochemistry 278: 147–155, 2005.

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Effect of hairpin-TFO on Effect of hairpin-TFO on transcriptiontranscription

The hairpin-TFO on transcription:

– of STE6 and CBT1

– An over producer of GAL4 protein was used

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The cellsThe cellsgrown in medium were induced with galactosetransfected with 1.5μM hairpin-TFO in the presence

of 0.8nM PEI PEI:

– to aid in transfection – to increase the stability of the triplex structure in vitro

The efficiency of transfection under these conditions was measured: – using pGAD424 plasmid After transfection

The cells were harvested at different time RNA was extracted RNA: subjected to RT-PCR in multiplex

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The sequence of the hairpin-TFO and a potential interaction The sequence of the hairpin-TFO and a potential interaction

of the hairpin TFO, with the target duplex and GAL4 proteinof the hairpin TFO, with the target duplex and GAL4 protein

The 65mer hairpin-TFO

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Optimization of RT-PCR Optimization of RT-PCR ACT1 gene contains two stretches of poly(pu/py) sequenceButnone of these have any complementarity to the poly(pu/py)sequence present upstream of STE6 and CBT1 genes.

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ACT1 geneACT1 gene

The gene should contain poly(pu/py) sequence

In the upstream region

But not similar to that in the upstream region of STE6 and CBT1

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Optimization of RT-PCR:Optimization of RT-PCR: Conditions Concentration of the primers for Conditions Concentration of the primers for

ACT1 and STE6 are variedACT1 and STE6 are varied

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Effect of transfection of hairpin-TFO on transcription of targeted genes of yeast strain Sc340 (A) STE6 transcripts measured by RT-PCR at different time points after transfection (B) CBT1 transcript levels

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The possible transcription complex recruited by the hairpin-TFO:DNA binding domain/ Activating domain of Gal4 Protein

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The reasonThe reasonfor the lower level of for the lower level of activation of STE6 activation of STE6

genegene

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The sequence of the hairpin-TFO and a potential interaction The sequence of the hairpin-TFO and a potential interaction

of the hairpin TFO, with the target duplex and GAL4 proteinof the hairpin TFO, with the target duplex and GAL4 protein

The 65mer hairpin-TFO

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The reasonThe reasonfor the lower level of activation of for the lower level of activation of

STE6 gene STE6 gene STE6 gene:

– the criteria for optimum distance of GAL4 recruitment is fulfilled

In the case of CBT1:

– the distance of the GAL4 recruitment site is more than what is suggested as the optimum distance.

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The lack of activation in a GAL4 The lack of activation in a GAL4 mutant: mutant: Down activation of gene expressionDown activation of gene expression

Activation through hairpin-TFO is Activation through hairpin-TFO is specifically mediated by GAL4specifically mediated by GAL4 proteinprotein

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Effect of transfection of hairpin-TFO on transcription Effect of transfection of hairpin-TFO on transcription of targeted genes in the yeast strain HF7c (GAL4−)of targeted genes in the yeast strain HF7c (GAL4−)

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TFOTFO as an anti tumor as an anti tumor Triplex DNATriplex DNA

A target for DNA-binding polycyclic A target for DNA-binding polycyclic acridine derivativesacridine derivatives

promise of therapeutic utility

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Antigene therapiesAntigene therapies

It’s based on the recognition and binding of a single oligonucleotide strand

To a double-stranded sequenceForming a triple helix

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Triplex DNA formationTriplex DNA formation A relatively weak and temporary

phenomenon

Therefore, molecules that selectively bind to and stabilize triple helices may show a variety of novel biological effects.

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Compounds: Polycyclic acridinesCompounds: Polycyclic acridines

A series of antitumor That bind to triplex DNA Whose synthesis has been previously

reported Have been tested for their interaction

with both purine and pyrimidine type triple helices

As a pyrimidine triplex model Antitumor activity

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Only Only purinepurine TFOs have been TFOs have been shown to mediate genome shown to mediate genome

modification without the need for a modification without the need for a targeted DNA-adducttargeted DNA-adduct

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TFOsTFOs For altering gene function

By either repressing transcription

Inhibiting DNA replication

Inducing site-specific mutagenesis and recombination

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DNA:RNA:DNA Triplex DNA:RNA:DNA Triplex FormationFormation

Their potential as tools in molecular biology

Therapeutic agents Unstable DNA:RNA triplexes play key

roles in many biological processes Inhibition of RNAse, DNAse I, and RNA

polymerase

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Models of structures that may mediate mRNA synthesis and DNA replication inhibition

by Triplex